1. Field of the Invention
The present invention relates to redox-responsive fluorogenic probes and liposome preparations comprising them, and to methods for detection and determination of vitamin C employing the same. Specifically, the present invention relates to a fluorogenic probe consisting of a porphyrin or phthalocyanine with a nitroxide radical(s), and especially a (phthalocyaninato)silicon complex with a nitroxide radical(s) as an axial ligand(s), to a liposome preparation comprising it, and to in vivo and in vitro methods for detection and determination of vitamin C using the same.
2. Description of Related Art
It is well known that vitamin C (ascorbic acid) is a water-soluble vitamin that is widely distributed in the plant kingdom, being particularly abundant in fruits and in green and yellow vegetables. Vitamin C plays important roles in body functions including (1) amino acid biosynthesis, (2) hormone secretion from the adrenal gland, and (3) synthesis of L-carnitine, a carrier that transports fatty acids to the mitochondria. It is also necessary for production of collagen in the connective tissue, and vitamin C deficiency is manifested as scurvy symptoms (loose teeth, weakening of blood vessels, hemorrhaging from the skin, impaired wound recovery and immunological function and mild anemia).
Because humans cannot synthesize vitamin C in the body, they must take in their entire required amount from the external environment through food, for example. Vitamin C is also known to have powerful antioxidant activity. Therefore, it is often added to processed foods and health foods as an antioxidant vitamin or as an antioxidant food additive.
The HPLC method, hydrazine colorimetric method and indophenol method have been used in the prior art as methods for detecting and determining vitamin C in foods or biological samples. Other methods have also been reported, such as a method involving two reactions conducted in the same reaction system, namely a reaction in which reduced ascorbic acid and oxygen yield oxidized ascorbic acid and hydrogen peroxide in the presence of ascorbate oxidase and a reaction in which the generated hydrogen peroxide and a chromogen are reacted in the presence of a peroxidase to produce pigments, and the ascorbic acid in the sample is determined based on the produced pigments (see Japanese Patent No. 4073963, for example), or a method wherein o-phenylenediamine is added to a vitamin C-containing sample and the sample is irradiated with polarized excitation light, the degree of polarization of the produced fluorescence is measured, and the quantity of vitamin C is determined based on the measured value (see Japanese Unexamined Patent Publication No. H11-326207, for example). These determination methods, however, still have problems such as complicated procedures (including those for pretreatment), needs of long periods for quantification, and/or low precision.
In recent years, the link between vitamin C and aging has been a focus of attention while high dosage treatment of vitamin C has also been reported to be effective for treatment of cancer. For example, an article on cancer treatment published in 2005, entitled “Pharmacologic ascorbic acid concentrations selectively kill cancer cells” has received interest, and results have been reported indicating that drip infusion of high concentration vitamin C is effective for cancer treatment (see Qi chen et al., Proc. Natl. Acad. Sci. USA 102 (38), 13604-13609 (2005), for example). However, the precise behavior of vitamin C in vivo has not been fully elucidated, and bioimaging and related technologies are highly expected to shed light on its in vivo function. Yet none of the methods mentioned above can be applied for bioimaging of vitamin C.
Methods applicable to bioimaging would be detection and determination of vitamin C by luminescence, such as a method using a dual molecule comprising a fluorescent chromophoric group such as dansyl or pyryl and a nitroxide radical, as a fluorogenic probe (see E. Lozinsky et al., J. Biochem. Biophys. Methods 38, 29-42 (1999), for example). Such fluorogenic probes, however, have a wavelength range of low permeability to biological tissue (≦650 nm) for both their excitation light and fluorescence, and are therefore poorly applicable for fluorescent bioimaging.